Our recent work on protein glycosylation has received attention from http://acceleratingscience.com. Following the full text.
Protein recovered from poultry and fish by-products is becoming more important as an ingredient in the food industry. Unfortunately, it is often degraded during the recovery process, reducing quality and usability. However, researchers are developing effective methods to improve recovered protein quality.
One of these techniques, based on the Maillard reaction, modifies proteins by covalently attaching carbohydrates under dry heat conditions. Unfortunately, although the process is effective, it is also slow, difficult to control and can result in formation of known mutagens such as advanced glycation end products (AGEs). These AGEs can develop during storage, even after the initial Maillard reaction has been stopped.
Instead of the dry process to glycate proteins, Hyrnets et al.1 investigated an aqueous state reaction to enzymatically add the amino sugar glucosamine (GlcN) to the protein. The reaction is catalysed by transglutaminase(TGase), an enzyme widely present in a many body tissues. This reaction takes place quickly at lower temperatures and is easier to control, producing a higher quality end product that is more stable during storage.
First the researchers extracted their protein substrate, natural actomyosin (NAM), from chicken breast muscle before incubating it with glucosamine. They performed the reaction under various temperatures and sugar concentrations to discover optimal conditions for the process. Once processed, the quality of the glycosylated protein end-products was assessed, and the research team used proteomic analysis techniques to determine the success and completeness of the enzymatic reaction.
Firstly, Hyrnets and co-workers evaluated glycoconjugation using matrix-assisted laser absorption/ionization (MALDI) – tandem time-of-flight (TOF/TOF) mass spectrometry to estimate how many GlcN residues had linked covalently with NAM. The optimal reaction environment for maximal binding was 37°C with a 1:1 ratio of sugars to protein; 15% of the peptides available were bound. The research team also used this assay to examine how TGase glycosylation compared to and competed with glycation by the Maillard reaction. At lower reaction temperatures of 25°C, glycation by the Maillard reaction was reduced in the presence of TGase, resulting in higher levels of the more stable glucosamine-actomyosin bond. This is beneficial during storage as Maillard reaction glycoconjugates are not so stable and continue to form AGEs.
Hyrnets et al. used liquid chromatography-mass spectrometry (LC-MS) to determine the actual site of enzymatic glycosylation on the proteins. Glycosylation catalyzed by TGase occurs at specific sites in the peptide sequence, so by comparing control with glycosylated samples, researchers could accurately define where and how the conjugation was occurring.
The researchers used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins, then digested gel regions of interest with trypsin. Samples were analyzed by nanoflow LC (Thermo Scientific) coupled to an LTQ XL-Orbitrap hybrid MS (Thermo Scientific). Peptide fragments were identified in the resulting MS data using the Proteome Discoverer 1.3 (Thermo Scientific) and the Uniprot protein database (SEQUEST, Thermo Scientific). LC-MS results confirmed that covalent binding of glucosamine with NAM was indeed due to TGase activity.
The researchers also measured quality of end product to show how enzymatic glycosylation performed compared with traditional protein modification. They found that solubility, emulsifying ability, and thermodynamic stability all increased in the glycosylated products, suggesting that the enzymatic glycosylation process could improve the additive ingredient making it more acceptable to the consumer.
1. Hyrnets, Y., M. Ndagijimana and M. Betti 2014. “Transglutaminase-catalyzed glycosylation of natural actomyosin (NAM) using glucosamine as amine donor: Functionality and gel microstructure,“Food Hydrocolloids 36 (pp. 26-36)”